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Use of recombinantly produced 15N3-labelled nicotianamine for fast and sensitive stable isotope dilution ultra-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry

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Abstract

Nicotianamine (NA) is an important metal chelator, implicated in the intra- and intercellular trafficking of several transition metal ions in plants. To decipher its roles in physiological processes such as micronutrient acquisition, distribution or storage, fast and sensitive analytical techniques for quantification of this non-proteinogenic amino acid will be required. The use of a recombinant Schizosaccharomyces pombe strain expressing a nicotianamine synthase (NAS) gene allowed for the production of [15N3]-NA, which was enriched from cell extracts through cation exchange and used for stable isotope dilution analysis of NA. Such an approach should be widely applicable to important bioanalytes that are difficult to synthesize. The analytical procedure comprises mild aqueous extraction and rapid Fmoc derivatization, followed by fast separation using ultra-performance liquid chromatography (UPLC) and sensitive detection by positive ion electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) with a chromatographic cycle time of only 8 min. Derivatization was optimized with respect to incubation time and species suitable for quantification. The limit of detection was 0.14 to 0.23 pmol in biological matrices with the response being linear up to 42 pmol. Recovery rates were between 83% and 104% in various biological matrices including fission yeast cells, fungal mycelium, plant leaves and roots.

15N3-labelled nicotianamine internal standard is synthesized by fission yeast cells growing on 15N-containing medium and expressing a nicotianamine synthase gene (NAS).

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Acknowledgements

This work was financially supported by the Deutsche Forschungsgemeinschaft (Cl 152/3-2) and in part by the European Union through its Sixth Framework Program for RTD (contract no FOOD-CT-2006-016,253).

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Author notes

  1. Aleksandra Trampczynska

    Present address: Bayer CropScience, 02-326, Warsaw, Poland

Authors and Affiliations

  1. Department of Plant Physiology, University of Bayreuth, Universitätsstrasse 30, 95440, Bayreuth, Germany

    Holger Schmidt, Aleksandra Trampczynska & Stephan Clemens

  2. Leibniz Institute of Plant Biochemistry, Department of Stress and Developmental Biology, 06120, Halle/Saale, Germany

    Christoph Böttcher

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  1. Holger Schmidt
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  2. Christoph Böttcher
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  3. Aleksandra Trampczynska
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  4. Stephan Clemens
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Correspondence to Stephan Clemens.

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Schmidt, H., Böttcher, C., Trampczynska, A. et al. Use of recombinantly produced 15N3-labelled nicotianamine for fast and sensitive stable isotope dilution ultra-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry. Anal Bioanal Chem 399, 1355–1361 (2011). https://doi.org/10.1007/s00216-010-4436-7

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  • DOI: https://doi.org/10.1007/s00216-010-4436-7

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